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Biochimie 93 (2011) 1701e1709

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Biochimie

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Research paper Substrate specificity of -related peptidase 13 activated by salts or glycosaminoglycans and a search for natural substrate candidates

Douglas Andrade a, Diego M. Assis a, Jorge A.N. Santos a, Fabiana M. Alves a, Izaura Y. Hirata a, Mariana S. Araujo b, Sachiko I. Blaber c, Michael Blaber c, Maria A. Juliano a, Luiz Juliano a,*

a Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-020 São Paulo, Brazil b Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil c Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306-4300, USA

article info abstract

Article history: KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary Received 28 April 2011 glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and Accepted 31 May 2011 its substrate specificity by employing a series of five substrates derived from the fluorescence resonance Available online 12 June 2011 energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic 0 residues with highest efficiency for R; furthermore, the S3 to S2 subsites accepted most of the natural Keywords: amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight Kinin peptide substrates were identified containing sequences of found in testis and one with myelin Protease Histatins basic sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides Kosmotropic salts present in higher primates with broad antifungal and mucosal healing activities that are generated from Myelin basic protein the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclu- sively at R25 (DSHAKRHHGYKRKFHEKHHSHRGYR25YSNYLYDN) that is the first cleavage observed inside the salivary gland. In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs. Ó 2011 Elsevier Masson SAS. Open access under the Elsevier OA license.

1. Introduction preferences in glandular epithelia, tonsils, esophagus, trachea, testis, salivary glands, vagina and cervix [8e10]. The physiological The tissue kallikrein members represent a cluster of 15 serine roles of KLK13 remain unknown, in contrast to KLK1 that is peptidases of -like S1A family clan PA(S) localized in involved in kinin release [11]; KLK3 hydrolyzes seminogelin I and II the human locus 19q13.4, and present a high degree in semen and contributes to its liquefaction after ejaculation of identity [1e7]. Human kallikrein-related peptidase 13 (KLK13) is [12,13]; KLK4 in prostate activates KLK3 [14,15], is modulated by one of these peptidases that is expressed in many tissues with a specific zinc [16] and is involved in dental enamel formation [17]; KLK6 activates PAR2 and cleaves glutamatergic receptors and myelin basic protein [18e22]; KLK5 and KLK7 are Abbreviations: Q-EDDP, (glutamyl-N-[ethylenediamine] 2,4-dinitrophenyl); Abz, involved in skin desquamation (for a recent review see [23]) and ortho-aminobenzoic acid; HOBt, N hydroxybenzotriazole; TBTU, 2-(1H-benzo- KLK8 participates in human epidermis and sweat in a proteolytic fl triazole-1-yl)-1,1,3,3-tetramethylaminium tetra uorborate; NMM, N-methyl-mor- cascade contributing to the skin barrier [24]. The associations of pholine; (PMC), 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Trt, trityl; DIPEA, diisopropylethylamine; TFA, Trifluor acetic acid; EDT, ethanodithiol; DMF, dime- KLK13 with some pathologies were earlier reported, and include thylformamide; NMM, 4-methylmorpholine; DCM, dichloromethane; GAGs, psoriasis vulgaris and atopic dermatitis [25] (where the peptidase is glycosaminoglycans; FRET, fluorescence resonance energy transfer; LMWK, low expressed in significant amounts), it is observed to be a favorable molecular weight kininogen; MCA, methyl coumarin amide; MALDI-TOF, Matrix- prognosis marker in ovarian cancer [26], is expressed in high levels assisted laser desorption/ionization-Time of flight; LCMS, liquid chromatography/ in some types of salivary gland tumors [27], is down regulated mass spectrometry. * Corresponding author. Tel.: þ55 11 5576 4450; fax: þ55 11 5575 9617. in testicular cancer [28,29] and is also reported to degrade the E-mail address: [email protected] (L. Juliano). extracellular matrix [30].

0300-9084 Ó 2011 Elsevier Masson SAS. Open access under the Elsevier OA license. doi:10.1016/j.biochi.2011.05.037 1702 D. Andrade et al. / Biochimie 93 (2011) 1701e1709

Detailed analyses of substrate specificity of peptidases are essen- We further tried to find other possible candidates as natural tial to identify their roles in mammalian organisms, to aid in the substrates for KLK13 using a support-bound FRET peptide library development of selective assays, and to guide the design of novel prepared on PEGA resin (a beaded polyethylene glycol dimethyla- inhibitors. The substrate specificity of KLK13 was earlier investigated crylamide copolymer) by the process of split-combine synthesis, by screening of fluorogenic 7-amino-4-carbamoylcoumarin positional which results in a single peptide sequence on each resin bead scanning-synthetic combinatorial libraries modified at the P1 to P4 [43e46]. The FRET peptide bound to a resin bead when hydrolyzed positions [31] andKLK13wasobservedtohaveselectivityoftheS1 by the peptidase turned the bead fluorescent; such beads were subsite for R, preference for hydrophobic amino acids by the S2 and S4 manually picked up and the remained peptide in the bead was subsites, and S3 selectivity mainly for R (Schechter and Berger sequenced by Edman degradation methodology. The identified nomenclature [32]). Though these synthetic combinatorial libraries peptides were synthesized and the cleavage sites by KLK13 were have provided valuable information about the specificity of numerous subsequently determined in solution and the kinetic parameters for peptidases [33,34] they are restricted to the non-prime side of the their hydrolysis were evaluated. peptidases, its application can be impaired for peptidases where the requires extended prime site substrates. In addition, 2. Materials and methods cleavages of these substrates other than at the amino site of the 7- amino-4-methylcoumarin group (peptidyl-Y-AMC)arenotdetected 2.1. Recombinant KLK13 with this library. Glycosaminoglycans (GAGs) and kosmotropic salts such as Mature KLK13 was expressed and purified from a baculovirus/ sodium citrate and sodium sulfate were reported to strongly acti- insect cell line system as previously described [22].Briefly, the KLK13 vate the hydrolytic activities of KLK3 and KLK6 [18,35,36] in which encodes for the mature form of KLK13 was inserted in contrast to KLK1 that is inhibited by salts [37]. In the present work frame, C-terminal of an enterokinase propeptide. To facilitate the we examined the modulation of salts and GAGs on hydrolytic purification procedure, a 6 His-tag was also included on the N- 0 activity of KLK13 and its S3 to S2 subsite substrate specificity taking terminal side of the enterokinase propeptide. The purification took as reference the fluorescence resonance energy transfer (FRET) advantage of the His-tag by Ni-NTA affinity chromatography. After peptide Abz-KLRSSKQ-EDDnp (Abz ¼ ortho-aminobenzoic acid and purification, KLK13 was activated by extended (i.e. 24 h) incubation EDDnp ¼ N-[2,4-dinitrophenyl]-ethylenediamine), which was with enterokinase at 1:100 ratio of enterokinase:pro-KLK13 (w/w). designed based on previously reported subsite requirements of Hydrolysis of the enterokinase propeptide proceeded to apparent KLK1 and KLK6 [18]. Five series of analogs of this peptide were completion and the released propeptide and enterokinase were synthesized with substitution of each amino acid, except Gln, and removed from the mature KLK13 by size exclusion chromatography. assayed as substrates of KLK13. The final yield of purified mature KLK13 was typically 20e25 mg per Histatins are a class of salivary peptides present in higher liter of culture. The final purity of mature KLK13 was assessed by primates, rich in basic amino acids [38] and have broad antifungal Coomassie Brilliant Blue staining of a 15% SDS-PAGE gel, N-terminal activity [39e41]. The systematic search by tandem mass spectros- sequencing, and mass spectrometry, and yielded a purity of at least copy of human saliva showed that the generation of histatin 3- 98% with no visible degradation (Fig. 1A) and the correct mature N- related peptides resulted mainly from the action of -like terminus. The purified, mature form of KLK13 remained a single chain proteases, and the first cleavage being at R25 (DSHAKRHHGYKRKF- and migrated as closely-spaced doublet bands under both reducing HEKHHSHRGYR25YSNYLYDN) [42]. The selectivity of KLK13 for and non-reducing conditions. The results of N-terminal sequencing hydrolysis at R and K and its co-localization with histatins in salivary revealed that both bands had identical termini consistent with glands [10] prompted us to examined the hydrolytic activity of mature KLK13 (VLNTXGTSGFLP). The 5th cycle of the reaction did not KLK13 on synthetic histatin 3 that is mainly processed in human give an amino acid assignment, and this is presumably due to saliva into histatin 6 (DSHAKRHHGYKRKFHEKHHSHRGYR25) and glycosylation of Asn. The doublet bands were separated by Phe- 25 then to histatin 5 by removing of C-terminal R [42]. Due to nomenex C4 reverse phase HPLC (Fig. 1-B) and analyzed by MALDI- selectivity of KLK13 to cleave at basic amino acids, the kininogenase TOF mass spectrometry (Fig. 1-C), and the peak of each fragment activity was also evaluated using low molecular weight (LMW) presented molecular masses 29,213 and 30,270 Da, respectively. kininogen, and a synthetic human kininogen fragment, Abz-MIS- These values are higher than the calculated mass of un-modified LMKRPPGFSPFRSSRI-NH2 that contains a bradykinin sequence. KLK13 presumably due to the result of heterogeneous glycosylation

Fig. 1. (A) Purified recombinant KLK13 analysis by 15% SDS-PAGE in reducing conditions. Lanes: 1-Molecular weight marker; 2-Mature form of KLK13 with doublet fragments; 3- and 4-Mature forms of KLK13 isolated by C4. (B) C4 reverse phase HPLC separation of doublet fragment. (C) MALDI-TOF analysis of KLK13 fragments. D. Andrade et al. / Biochimie 93 (2011) 1701e1709 1703

(with the higher-mass form containing approximately 6 additional was at 38 C) with 0.5 mM heparin or 1.5 M sodium citrate. The hexose units). Both fragments exhibited similar enzymatic activity were pre-incubated in the assay buffer for 3 min before against Tos-GPR-AMC. Molar concentration of active KLK13 was the addition of substrate. Fluorescence changes were monitored determined by titration with MUGB (4-Methylumbelliferyl p-guani- continuously at lex ¼ 320 nm and lem ¼ 420 nm. When fluorogenic dinobenzoate hydrochloride) by spectrofluorimetric tritation as MCA peptides were used, the excitation and emission wavelengths previously described [47]. Bovine myelin basic protein was purchased were changed to lex ¼ 380 and lem ¼ 460 nm, respectively. The from (AbD Serotec, Raleigh, NC, USA). concentrations for initial rate determinations were chosen at a level intended to hydrolyze less than 5% of the added substrate 2.2. Peptide synthesis over the time course of data collection. The slope of the generated fluorescence signal was converted into micromoles of substrate All FRET peptides and histatin-3 were obtained by solid-phase hydrolyzed per minute based on a calibration curve obtained from peptide synthesis as previously described [48,49] and using the the complete hydrolysis of each peptide. Fmoc-procedure in an automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu,- 2.6. Support-bound FRET peptide library screening Tokyo, Japan). The peptides were synthesized in TGR-resin (loading 0.2 mmol/g) using HBTU/HOBt as coupling reagent and the cleavage For all assays, the library beads were washed with water (3) of peptide-resin was accomplished with TFA:anisol:EDT:water and the assay buffer (3) before the addition of the enzyme. The (85:5:3:7). All peptides obtained were purified by semi-preparative reactions were stopped by dilution with 3 M HCl, and the mixtures HPLC on an Econosil C-18 column. The molecular weight and purity were washed thoroughly until pH 5e6 was reached. The beads (94% or higher) of synthesized peptides were checked by amino acid were transferred to a glass dish and inspected by fluorescence analysis and MALDI-TOF mass spectrometry, using a Microflex e LT microscopy (Stereomicroscope Stemi-Zeiss), and the fluorescent mass spectrometer (Bruker e Daltonics, Billerica, MA, USA). Stock beads were collected and transferred to a TFA-treated cartridge solutions of the peptides were prepared in DMF and the concen- filter for on-resin sequence analysis. The amino acid sequence and trations were measured spectrophotometrically using the molar cleavage point were determined by Edman degradation using extinction coefficient of 17,300 M 1 cm 1 at 365 nm. a PPSQ/23 protein sequencer (Shimadzu, Japan). KLK13 was assayed as follows: 20 mg of resin in 50 mM Tris, 1 mM EDTA, pH 7.5, at 2.3. Synthesis of support-bound FRET peptide library 25 C for 5 h with 75 nM of enzyme. The identified peptide sequences susceptible to hydrolysis by KLK13 were synthesized as The syntheses of libraries were carried out manually as previ- FRET Abz-peptidyl-Q-EDDnp peptides and assayed in solution with ously described [46]. Briefly, the libraries were synthesized on 1 g of the enzyme. PEGA1900 resin [50] in a 20 column Teflon synthesis block, using protected Fmoc amino acids. The resin was evenly distributed in the 2.7. Digestion of myelin basic Protein by KLK13 20 wells of the Teflon synthesis block, and the Fmoc groups were removed. Prior to coupling the Fmoc amino acids (1 equiv.) were Bovine myelin basic protein (MBP) was added to KLK13 at pre-activated with HOBt (1 equiv.), TBTU (1 equiv.) and NMM a 2000:1 mass ratio in 50 mM Tris and 1 mM EDTA, pH 7.5. This (2 equiv.) in DMF (1 ml) for 6 min and the activated amino acids mixture was incubated at 35 C, and time points were taken at 2, 4, were added, one to each of the 20 wells. After the completion of the 8, and 12 h. The MBP and degradative fragments were resolved coupling, the block was filled with DMF up to 1 cm above the top of using Tricine SDS-PAGE (16.5%) and developed by Comassie-Blue the wells and inverted, and the resin was mixed vigorously by and two bands were isolated and the N-terminal sequenced by agitation for 30 min in the mixing chamber. The block was again Edman degradation to identify the cleavage sites. inverted, evenly distributing the resins into the wells for washing and removal of the Fmoc group. This procedure was repeated for 2.8. Kininogenase activity of KLK13 the incorporation of all the randomized positions. After the randomized positions, the Fmoc-K(Abz-Boc) and Fmoc-K(Dnp) Low molecular weight human kininogen (LK) was purchased were incorporated. The side chain protecting groups were from Calbiochem. The ability of KLK13 to cleave the human LK was removed by treatment with a mixture of TFA:thioanisole:ethane- evaluated incubating 1 ml of LK (100 mg/ml) and KLK13 (1.8 nM) in dithiol:water (87:5:5:3) for 8 h. The resin was washed with 95% reaction mixtures containing 50 mM Tris, 0e1.5 M sodium citrate, acetic acid (4), DMF (4), 5% DIPEA in DMF (3), DMF (3), DCM pH 7.5, at 35 C for 4 h. Ethanol (3:1, v/v) was added, and the (6) and dried under vacuum. mixture was centrifuged at 1000 g for 15 min. The kinin content in the supernatant was measured by radioimmunoassay, as previ- 2.4. Glycosaminoglycans ously described [52] KLK1 was also assayed as comparative control.

Size-defined (12,000 Da) bovine lung heparin (The Upjohn Co.) 3. Results was prepared by using a size exclusion column approach [51]; heparan sulfate (16,000 Da) from bovine lung was a generous gift 3.1. Conditions for high KLK13 activity from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy); dermatan sulfate (12,000 Da) and chondroitin sulfate The FRET peptide Abz-KLRSSKQ-EDDnp was taken as the initial (25,000 Da) were purchased from Seikagaku Kogyo Co. (Tokyo, substrate to search for the best conditions required for efficient KLK13 Japan). hydrolytic activity. The peptide Abz-KLRSSKQ-EDDnp was hydrolyzed only at the ReS bond, and this activity of KLK13 at pH 7.5 was highly 2.5. Kinetic measurements activated by sodium citrate or sodium sulfate, as shown in Fig. 2A. High activation was also observed with heparin up to 0.5 mM(Fig. 2-B) The FRET peptides were assayed in a Shimadzu RF-1501 spec- and chondroitin and dermatan sulfate also activated KLK13 but at trofluorometer, at 35 C. The assays were performed in 50 mM Tris, concentration range 20e40 mM (data not showed). KLK13 bound 1 mM EDTA, pH 7.5 and 35 C (the temperature of highest activity to heparineSepharose resin and was eluted with 100 mM NaCl, 1704 D. Andrade et al. / Biochimie 93 (2011) 1701e1709

Fig. 2. (A) Activation of KLK13 by sodium citrate (C) and sodium sulfate (,). (B) Activation of KLK13 by heparin (C) pH profiles of KLK13 activity in the presence of sodium citrate (:), heparin (-) and only with buffer (C). The reactions were performed in 50 mM Tris, 1 mM EDTA, pH 6.5e9.0, using as substrate 7.5 mM Abz-KLRSSKQ-EDDnp, and [E] ¼ 3.8 nmol at 35 C. indicating a significant favorable electrostatic interaction between has a particular preference for K in contrast to R, as indicated by the heparin and KLK13 that was not observed with KLK1 and KLK6. The kcat/Km value for the hydrolysis of the peptide Abz-KLRYKSKQ- pH profiles of KLK13 activities in the presence of 1 M sodium citrate EDDnp that is 170-times higher than that of the peptide Abz- and heparin are showed in Fig. 2-C. The KLK13 activity was reduced KLRYRSKQ-EDDnp. The second best substrate of this series contains 0 close to zero in the presence of 50 mM Tris HCl (with no sodium N and the peptides with F, S and H at the P1 position were still citrate or heparin). The presence of NaCl, KCl, MgCl2 and CaCl2 up to efficiently hydrolyzed. Not unexpectedly, the peptide with X ¼ Pat 0 1 M concentration did not activate the enzyme. the S1 subsite was completely resistant to hydrolysis due to the imide bond of P. The kinetic parameters for hydrolysis of the series 0 Abz-KLRSXKQ-EDDnp (Table 3) also show that the S2 subsite of 3.2. Substrate specificity of KLK13 using FRET peptides derived KLK13 does not have restricted specificity but does present pref- from Abz-KLRSSKQ-EDDnp erence for the basic amino acid R, and the resistance to hydrolysis of 0 the peptide Abz-KLRSEKQ-EDDnp indicates that the S2 of KLK13 The peptide Abz-KLRSSKQ-EDDnp was taken as reference, and does not accepted negative charge. a series of five related FRET peptides were synthesized and assayed as substrates for KLK13 in 50 mM Tris at pH 7.5 and 35 Cinthe presence of 0.5 mM heparin, and for some peptides the assays were 3.3. Screening of support-bound FRET peptide library done in the presence of 1.5 M sodium citrate. The specificity of the S1 subsite was explored with the peptide series Abz-KLXSSKQ-EDDnp The peptides selected from the screening of this library were (where X ¼ R, K, F, Y, M, L, V, W, D, E, G, H, I, N, P, Q, S, and T). Only the synthesized as FRET Abz-peptidyl-Q-EDDnp peptides and assayed peptides Abz-KLRYSSKQ-EDDnp and Abz-KLKYSSKQ-EDDnp (i.e. with KLK13. Their sequences and velocities of hydrolysis in the X ¼ RorX¼ K) were hydrolyzed and only at the peptide bonds ReS presence of 1 M sodium citrate are shown in Table 4. The suscep- and KeS, respectively. The kinetic parameters of these hydrolyses in tibility to hydrolysis by KLK13 of the selected peptides was higher the presence of heparin and sodium citrate at pH 7.5 are presented in the presence of sodium citrate than in the presence of 50 mM e in Table 1. All other peptides were resistant in both conditions up to Tris HCl, and most of these peptides were cleaved at the carboxyl side of R. Only two peptides were cleaved at K and each with lower 60 nM of KLK13. These results indicate that the S1 subsite of KLK13 has a very restricted specificity for basic amino acids. The resistance velocities. It is noteworthy that the basic amino acids have signif- to hydrolysis of peptide Abz-KLNSSKQ-EDDnp contrasts with the icant frequency in all positions besides the P1 position and eight out of twenty sequences were found in proteins from testis. reported hydrolysis of peptides containing N at the P1 position in the screening of fluorogenic 7-amino-4-carbamoylcoumarin positional One human myelin basic protein sequence (GPVKKRNM) was scanning-synthetic combinatorial libraries [31]. found as a potential substrate of KLK13 in the support-bound fi The FRET peptides in the series Abz-XLRSSKQ-EDDnp, Abz- peptide library that was con rmed by the hydrolysis of the FRET KXRSSKQ-EDDnp, Abz-KLRXSKQ-EDDnp and Abz-KLRSXKQ-EDDnp, were synthesized in order to explore the specificity of the subsites 0 0 Table 1 S3,S2,S1 and S2 , respectively. The kinetic parameters for their Kinetic parameters for the hydrolysis by KLK13 of the reference peptides Abz- hydrolysis by KLK13 are shown in Tables 2 and 3. All the peptides of KLRSSKQ-EDDnp and Abz-KLKSSKQ-EDDnp in the presence of heparin and sodium the four series were hydrolyzed only at the ReS (or ReX) bond. citrate. 1 1 The parameters for the hydrolysis of the series Abz-KXRSSKQ- Activator kcat (s) Km (mM) kcat/Km (mM s) EDDnp (Table 2) shows that the S2 subsite of KLK13 did not Abz-KLRYSSKQ-EDDnp present restricted specificity; however, higher kcat/Km values were 0.5 mM heparin 3.2 0.1 2.2 0.1 1455 80 observed with substrates containing basic or hydrophobic amino 1.5 M sodium citrate 3.4 0.2 1.2 0.05 2834 204 acids at the P2 position. Similarly, an S3 subsite preference for basic Y and hydrophobic residues was observed as demonstrate from the Abz-KLK SSKQ-EDDnp 0.5 mM heparin 0.50 0.03 31 216 3 parameters of hydrolysis of the series Abz-XLRSSKQ-EDDnp 1.5 M sodium citrate 0.10 0.01 5.3 0.3 19 2 (Table 2). However, the peptide with the negatively charged The reactions were performed in 50 mM Tris, 1 mM EDTA, pH 7.5, 0.5 mM heparin residue E at the P position was resistant to hydrolysis. 3 and [KLK13] ¼ 3e10 nmol at 35 C. The parameters and the standard errors were The kinetic parameters for the hydrolysis of the peptide series obtained from three determinations. The errors associated to k /K were obtained 0 cat m Abz-KLRXSKQ-EDDnp (Table 3) show that the S1 subsite of KLK13 by error propagation of the kcat and Km errors. D. Andrade et al. / Biochimie 93 (2011) 1701e1709 1705

Table 2 Table 3 Kinetic parameters for the hydrolysis by KLK13 of the series of peptides Abz- Kinetic parameters for the hydrolysis by KLK13 of the series of peptides Abz- KXRSSKQ-EDDnp and Abz-XLRSSKQ-EDDnp in the presence of heparin (i.e. evalu- KLRXSKQ-EDDnp and Abz-KLRSXKQ-EDDnp in the presence of heparin (i.e. evalu- 0 0 ating the specificity of S2 and S3 subsites, respectively). ating the specificity of S1 and S2 subsites, respectively).

1 1 1 1 X kcat (s) Km (mM) kcat/Km (Mm s) X kcat (s) Km (mM) kcat/Km (mM.s) 0 Abz-KXRSSKQ-EDDnp (P2) Abz-KLRXSKQ-EDDnp (P1 ) R# 5.60 0.03 1.74 0.03 3218 58 K 26.6 0.1 3.10 0.02 8581 64 (0.50 0.05) (0.74 0.02) (676 70) (0.55 0.01) (0.93 0.01) (591 12) I 8.84 0.04 2.96 0.05 3027 53 N 11.8 0.1 5.33 0.03 2224 23 K 8.96 0.05 4.74 0.03 1890 16 F 4.1 0.06 2.40 0.01 1708 26 L(ref) 3.22 0.02 2.28 0.03 1412 21 S(ref) 3.21 0.02 2.24 0.03 1433 21 P 7.56 0.07 5.23 0.01 1445 14 H 4.24 0.05 3.00 0.06 14,013 33 V 6.05 0.04 5.05 0.05 1200 12 Q 4.8 0.1 7.9 0.1 607 15 F 7.04 0.05 7.04 0.06 1000 11 A 2.46 0.01 4.06 0.05 606 8 E ee 860*10 I 0.60 0.01 1.35 0.02 444 10 Y 6.9 0.1 10.7 0.1 635 11 V 1.52 0.03 3.60 0.02 422 9 H 2.9 0.1 5.7 0.1 509 20 L 1.20 0.03 3.81 0.01 314 8 M ee 531*12 G 2.05 0.06 10.20 0.02 200 6 T 4.9 0.08 8.9 0.02 550 9 R 0.34 0.02 5.9 0.1 58 10 S 5.7 0.06 11.0 0.07 518 10 (3.07 0.05) (1.54 0.03) (2000 51) A 4.0 0.03 8.5 0.02 469 4 E e NH e Q 1.6 0.07 6.0 0.04 267 12 0 G 2.0 0.06 12.7 0.02 161 5 Abz-KLRSXKQ-EDDnp (P2 ) N 1.1 0.1 9.9 0.1 111 10 R 8.9 0.1 1.03 0.01 8640 128 L 29.5 0.1 7.60 0.03 3881 20

AbzeXLRSSKQ-EDDnp (P3) F 14.8 0.1 4.02 0.03 3681 37 R 6.45 0.05 3.92 0.04 1135 17 Q 17.0 0.1 8.30 0.04 2048 16 K(ref) 3.20 0.02 2.25 0.03 1422 20 A 6.9 0.05 3.83 0.02 1816 16 I 10.6 0.1 7.80 0.02 1359 13 S(ref) 3.2 0.02 2.20 0.03 1454 22 F 7.24 0.05 6.9 0.1 1049 17 V 6.9 0.03 7.02 0.05 982 8 V ee 800*25 H 2.9 0.07 3.45 0.03 840 24 H 6.32 0.03 8.0 0.1 790 11 N 4.5 0.07 6.00 0.05 750 13 A 6.06 0.04 10.3 0.1 588 7 G 3.3 0.02 5.34 0.07 618 9 Q 5.51 0.01 10.40 0.06 529 3 P 0.7 0.01 6.42 0.02 109 9 N 5.03 0.03 14.6 0.1 344 3 I 0.5 0.02 5.54 0.02 91 3 L 3.86 0.03 12.6 0.1 306 3 E e NH e E e NH e The reaction conditions were as in Table 2 and NH ¼ no hydrolysis. The values in The reactions were performed in 50 mM Tris, 1 mM EDTA, pH 7.5, 0.5 mM heparin parenthesis were obtained in the presence of 1 M sodium citrate. and [KLK13] ¼ 3e10 nmol at 35 C. The values in parenthesis were obtained in the presence of 1 M sodium citrate. Reference peptide Abz-KLRSSKQ-EDDnp (ref).

*These kcat/Km values were obtained in pseudo-first order conditions because the Km values were too high and impaired the kinetic parameter determination due to inner filter of fluorescence. #Two cleavages was observed with Abz-KR*RYSSKQ- 3.5. Kininogenase activity of KLK13 EDDnp, the cleavage indicated by Y was 80% and the cleavage indicated by * was 20%. NH ¼ no hydrolysis. The parameters and the standard errors were obtained Low molecular weight human kininogen (LK) incubated with from three determinations. The errors associated to kcat/Km were obtained by error KLK13 released a low amount of kinin compared with the activity of propagation of the k and K errors. cat m KLK1. This low kininogenase activity of KLK13 was dependent on the presence of sodium citrate as shown in Fig. 5. We also examined the KLK13 hydrolysis time course of the human kininogen fragment peptide Abz-GPVKKRNMQ-EDDnp as indicated in Table 4. This Abz-MISLMKRPPGFSPFRSSRI-NH2 which was rapidly cleaved at the finding and the detection of KLK13 in neurons and glial cells [8] ReS bond with release of Abz-MISLMKRPPGFSPFR-OH, and this prompted us to assay commercially available bovine myelin basic fragment was subsequently slowly hydrolyzed at the K-R bond with protein as a substrate for KLK13. Fig. 3 shows the SDS-PAGE of the low yield of bradykinin (Fig. 6). These results were confirmed by highly digested myelin basic protein and at least two cleavage sites KLK13 hydrolysis in the presence of 1 M sodium citrate of the fi . Y . . Y . 1 were indenti ed, namely YGGR ASDYK and PRHR DTGI peptide Abz-GFSPFRYSSRIQ-EDDnp (kcat ¼ 0.3 s , Km ¼ 0.6 mM and 1 1 kcat/Km ¼ 500 mM .s ) and by the very slow hydrolysis of the peptide Abz-MISLMKYRPQ-EDDnp. 3.4. Hydrolysis of histatin 3 by KLK13

Histatin 3 was synthesized Abz at the N-terminal end in order to 4. Discussion increase the detection of the peptide and its products of hydrolysis by HPLC analysis with fluorescence detector (Abz-DSHAKRHH- The hydrolytic activity of KLK13 is substantially activated by the GYKRKFHEKHHSHRGYR25SNYLYDN). The time course of its hydro- kosmotropic salts sodium citrate and sodium sulfate, and in their lysis by KLK13 in the presence of 0.5 mM heparin is shown in Fig. 4 absence the KLK13 activity was very low with most of the assayed with the HPLC profiles of the reaction mixture sampled at different peptide substrates. Although this observation may be puzzling, the times. The hydrolysis of histatin 3 occurred only at the R25eS also high activities of KLKs 3 and 6 in the presence of sodium citrate peptide bond, with a velocity ¼ 35 nmol/min/nmol of enzyme. It is suggest that this KLK may be effectively modulated by in vivo micro- noteworthy that even after 24 h of reaction only the R25eS peptide environment concentrations of ions. The macromolecular crowding bond was cleaved, which indicates a remarkable specificity of concept seems to be adequate to interpret the activation of the KLKs KLK13 for this segment of histatin 3. Similar results were obtained 3, 6 and 13 by high sodium citrate concentrations. The macromo- by the activation of KLK13 with 1 M sodium citrate (data not lecular crowding obtained by high salt concentration was reported shown). to compact KLK3 [35] with reduction of surface-to-volume ratio, 1706 D. Andrade et al. / Biochimie 93 (2011) 1701e1709

Table 4 Sequences and hydrolysis of FRET peptides obtained from support-bound peptide library.

Sequences Velocity of Hydrolysis Human protein containing the peptide sequence (with database access) Abz-KLRYSSKQ-EDDnp 130 Reference peptide Abz-HK*TRYSEAQ-EDDnp 120 c114 SLIT-like testicular protein isoform (XP_002344102.1) Abz-GPKQRYSR*RQ-EDDnp 50 Bromodomain adjacent to zinc finger domain protein 1A (Q9NRL2 (BAZ1A_human) Abz-EVK*RYKTYQ-EDDnp 50 Histone-lysine N-methyltransferase (Q8IZD2 MLL5_human) Abz-RQIRYKNEQ-EDDnp 40 Ankyrin repeat domain-containing protein (Q8N7Z5 ANR31_human) Abz-GPNLRYARQQ-EDDnp 30 1) Sperm-associated antigen 17 (Q6Q759.1jSPG17_human) 2) Cancer/testis antigen 75 (Q6PK30.2jCT75_human) Abz-GPKLYRRYIQ-EDDnp 20 Not found Abz-TMVK*QARYKQ-EDDnp 20 Neutrophil cytosolic factor 4 (Q15080 NCF4_human) Abz-GPVK*KRYNMQ-EDDnp 20 1) Spermatogenesis-associated protein (Q8NHS9jSPT22_human) 2) Myelin basic protein (MBP) (spjP02686jMBP_human) Abz-AMER*ARYMQ-EDDnp 20 Sarcoma antigen NY-SAR-22 (Q86WF6_ human) Abz-GPRMYRYKLQ-EDDnp 15 IQ motif containing GTPase activating protein (Q59HA3_ human) Abz-GPRYVGGRVQ-EDDnp 15 Testis-specific Y-encoded-like protein (Q86VY4.2jTSYL5_human) Abz-NKRYHPNAQ-EDDnp 15 Dual specificity testis-specific protein kinase 1 (Q15569 TESK1_human) Abz-GPVAK*NRYSQ-EDDnp 15 1) Testis-expressed sequence 2 protein (Q8IWB9jTEX2_human) 2) Spermatogenic leucine zipper protein 1 (Q9BXG8jSPZ1_human) Abz-HPRYAMR*RQ-EDDnp 10 1) T-complex-associated testis-expressed protein 1 (Q5JU00.1_human) 2) Cancer/testis antigen 42 (Q9BXT5.1jTEX15_human) Abz-GPFAYQRYHQ-EDDnp 10 Not found Abz-GPRYQVK*KFQ-EDDnp 10 Glutamate receptor 4 precursor (P48058jGRIA4_human) Abz-GPRYSK*EIQ-EDDnp 5 Not found Abz-NWR*ERYSLQ-EDDnp 5 Not found Abz-GPKYKYYSIAQ-EDDnp 5 Cancer/testis antigen 42 (Q9BXT5.1jTEX15_human) Abz-EPHMKYHQ-EDDnp 5 Neuropeptide Y receptor type (Q15761.1jNPY5R_human)

Conditions of hydrolysis: 50 mM Tris, pH 7.5, 35 C, [KLK13] ¼ 8.7 nM and a fixed concentration of 10 mM of each peptide. Y indicates the preferred cleavage site (at least 90%) and * indicates the a secondary cleavage site. The only exception was the peptide Abz-GPKYKYYSIAQ-EDDnp where both cleavage sites were equally hydrolyzed. Velocity unit ¼ pmol/min nmol E.

which results in an active conformation of the enzyme; for review to the presence of high concentration chondroitin sulfate in tonsils and concept explanation of macromolecular crowding see [53]. [58] and in saliva [59] that also activates KLK13 (data not showed). Sodium citrate is present in normal prostate tissue and seminal fluid The S1 subsite of KLK13 presented strict specificity for the basic in high concentration [54,55,56], and it can be a potential physio- amino acids R and K, with large preference for R compared to K as logical modulator of KLK13 activity since it is also present in pros- shown by the kcat/Km value for the cleavage of Abz-KLRYSSKQ- tate, testis and in female reproductive organs [10]. In addition, EDDnp that was two orders of magnitude higher than that of Abz- KLK13 was reported to activate KLK2 and KLK3 by cleaving the ReI KLKYSSKQ-EDDnp. These observations are in agreement with the bond in the common sequence .IQSRYIVGG. [57], that is in screening of the support-bound peptide library that selected only accordance to our observed KLK13 specificity, and then sodium peptides hydrolyzed at R or K residues (Table 4). The subsites S2,S3, 0 0 citrate can also modulate the activation cascade these in S1 and S2 of KLK13 also accepted basic amino acids as indicated prostate. The hydrolysis of histatin 3 exclusively at R25 (Fig. 4)by from kinetic parameters of hydrolysis of the FRET peptides series KLK13 activated by heparin seems to be a relevant observation due derived from Abz-KLRSSKQ-EDDnp. The efficient hydrolysis of the

Fig. 3. SDS-PAGE of myelin basic protein digested by KLK13 in 18% polyacrylamide gel, developed by Coomassie Blue. The hydrolysis was performed in 50 mM Tris, 1 mM EDTA, 0.5 mM heparin, pH 7.5 at 35 C. At least two cleavage sites were indentified: .YGGRYASDYK. and .PRHRYDTGI. by N-terminal sequencing using Edman degradation. D. Andrade et al. / Biochimie 93 (2011) 1701e1709 1707

Fig. 4. Hydrolysis of histatin 3 by KLK13 in the presence of heparin. Reaction conditions 50 mM Tris, 1 mM EDTA pH 7.5 [E] ¼ 13 nmol with 0.5 mM heparin. The substrate is 38 mM 25 Abz labeled histatin 3 (Abz-DSHAKRHHGYKRKFHEKHHSHRGYR YSNYLYDN-NH2). The products were identified by mass spectrometry.

peptides with basic amino acids at P2 position suggested that the sequence of myelin basic protein as a potential substrate of KLK13 commercial peptide Z-RR-MCA would be a convenient substrate for was confirmed by the efficient hydrolysis of bovine equivalent KLK13, however this peptide was cleaved at the ReR bond instead protein (Fig. 3). It is possible that KLK13 plays a role in the cleavage of at the R-MCA bond as detected by HPLC analysis of the hydrolysis of myelin basic protein because this kallikrein was found in neurons products. This result suggests that for a short peptide constituted of and glial cells by immunohistochemical technique [8]. In addition, a pair of R flanked by hydrophobic groups put the R residues at S1 glycosaminoglycans that activate KLK13 are associated with central 0 and S1 subsites of KLK13. nervous system remodeling, development, and disease (for reviews The identification of eight substrates of KLK13 in the screening see [60,61]). of the support-bound peptide library with sequences that are The co-localization of KLK13 and its activators in different present in proteins from testis appears to be a significant obser- tissues reported above incite questions about how the in vitro vation because only in testis were found five splice variants in conditions used in the present work would mimic physiological or addition to the normal KLK13. In testicular cancer only normal pathological conditions in vivo. Our data spanned a large range of KLK13 is expressed, while KLK13 variants are expressed only in concentration of the activators that possibly included those found normal testis [28,29]. At the moment it would be very speculative in the normal or pathological tissues. to correlate possible physiological or pathological roles of the The kininogenase activity of KLK13 on human LMW kininogen is hydrolysis by KLK13 of the testis proteins indicated in Table 4, very poor compared with that of KLK1, although KLK13 was able to however this finding merits further investigation. The identifica- digest it. As shown in the hydrolysis of the human kinogen frag- tion in the support-bound peptide library of a peptide with ment peptide Abz-MISLMKRPPGFSPFRSSRI-NH2 (Fig. 6) the low kininogenase activity is due to the resistance of hydrolysis of the KeR bond in the N-terminal side of the bradykinin sequence in the kininogen.

Fig. 5. Kininogenase activity of KLK13 towards human LMW kininogen in the presence of different concentrations of sodium citrate. Stoichiometric amounts (16 pmol) of Fig. 6. Time course of hydrolysis by KLK13 of the human kininogen fragment Abz-

KLK13 and kininogen were used in the absence or presence of citrate ions (0.1e1.25 M). MISLMKRPPGFSPFRSSRI-NH2. The substrate and their products of hydrolysis were The buffer system used was 50 mM Tris, pH 7.5 containing 1 mM EDTA. The reaction quantified by HPLC and are shown as percentage of product formation or substrate mixtures, in a final volume of 80 mL, were incubated for 4 h at 35 C. degradation. The products were identified by mass spectrometry. 1708 D. Andrade et al. / Biochimie 93 (2011) 1701e1709

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